转运siRNA的双靶向长循环多功能信封式纳米基因载体的研究

发布时间：2018-07-07 23:14

本文选题：肝靶向 + 多功能信封式纳米装置　；参考：《青岛大学》2015年硕士论文

【摘要】：本课题制备了一种转运si RNA的非病毒双靶向长循环多功能信封式纳米装置(Multifunction Envelop-type Nano Device,MEND)。MEND为复合纳米脂质体,同时具有脂质体和纳米粒的特性,通过对其脂质成分进行修饰,达到长循环和肝癌细胞双重靶向的目的,转染效率提高。修饰后的脂质分子结构为:甘草次酸(Glycyrrhetinic,GA)-聚乙二醇(Polyethylene glycol,PEG)-肽(Peptide,Pp,基质金属蛋白酶MMP的底物)-二油酰磷脂酰乙醇胺(1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine,DOPE)。各成分通过酰胺反应结合,核磁共振氢谱法和质谱法进行结构鉴定;PLL压缩的si RNA溶液水化脂质体薄膜后,得到si RNA-MEND。依次利用透射电镜、动态光散射法和超滤离心法,检测si RNA-MEND的形态、粒径电位以及包封率;体外酶解实验考察si RNA-MEND在血浆中的稳定性;采用MTT法和荧光标记法,考察si RNA-MEND对肝癌BEL-7402细胞生长抑制和转染效率;利用K-Ras-si RNA-MEND对BEL-7402细胞转染,考察细胞侵袭转移和基因沉默。结果表明修饰物GA-PEG-Pp-DOPE成功合成;si RNA-MEND粒子呈光滑的球形,具有明显的脂质双分子层和指纹结构,其粒径分布范围为163.5±20.0nm,平均电位为0.614±0.05m V;MEND对si RNA的包封率为82.8%。酶解实验表明MEND可以保护si RNA免受血浆降解长达120h,是裸si RNA的40倍;修饰成分中的肽可以被MMP-2特异性降解。MEND的细胞毒性小,在MEND转染下细胞的生长平均生长率为87.03±4.65%,si RNA-MEND可以高效率的转染进入细胞质。由K-Ras-si RNA-MEND转染的细胞,其侵袭转移能力明显下降,K-Ras蛋白的表达水平比未转染细胞降低78.61倍。MEND的制备工艺简单,对si RNA具有较强的保护作用,可以明显延长si RNA在血液循环中的时间,转染效率较高。
[Abstract]:In this paper, a novel multifunction envelope device (multifunction Envelop-type Nano DeviceMend), which transports siRNA, was prepared as a composite nano-liposome with the properties of liposome and nanoparticles. The efficiency of transfection was improved with the aim of double targeting of long circulation and hepatoma cells. The modified lipids were composed of glycyrrhetinic acid (GA) -polyethylene glycoline (PEG) -peptide (PeptidePp) and dioleoyl-sn-glycero-3-phosphoanolaminedoPE (1-dioleoyl-sn-glycero-3-phosphoanolamine). The structures of the liposome films were identified by hydrogen NMR and mass spectrometry. Si RNA-MEND was obtained after hydration of liposome membrane in the solution of si RNA compressed by PLL. The morphology, particle size potential and encapsulation efficiency of siRNA-MEND were detected by transmission electron microscopy, dynamic light scattering and ultrafiltration centrifugation. The stability of siRNA-MEND in plasma was investigated by enzymolysis in vitro. The growth inhibition and transfection efficiency of BEL-7402 cells were investigated by siRNA-MEND, and the invasion, metastasis and gene silencing of BEL-7402 cells were studied by K-Ras-si RNA-MEND transfection. The results showed that the modified GA-PEG-Pp-DOPE successfully synthesized simium-RNA-MEND particles with smooth globular shape, obvious lipid bimolecular layer and fingerprint structure. The particle size distribution range was 163.5 卤20.0nm.The average potential was 0.614 卤0.05m VnMEND and the encapsulation efficiency of siRNA-MEND to si RNA was 82.8 folds. The results of enzymatic hydrolysis showed that met could protect siRNA from degradation of plasma for 120 hours, 40 times as much as bare siRNA, and the peptides in modified components could be degraded by MMP-2 specifically. The average growth rate of the cells transfected with met was 87.03 卤4.65 and siRNA-MEND could transfect into the cytoplasm efficiently. The invasiveness and metastasis ability of the cells transfected with K-Ras-si RNA-MEND was reduced 78.61 times than that of untransfected cells. The time of si RNA in blood circulation was prolonged, and the transfection efficiency was higher.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R450

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